Compositions and methods for inducing cell dedifferentiation

ABSTRACT

The present invention provides 2,6-disubstituted purine compounds, compositions and methods for dedifferentiating lineage committed mammalian cells into stem cells. The present invention also provides methods of inducing dedifferentiation of lineage committed mammalian cells into stem cells, which can be further differentiated into various lineage committed cells. Methods of identifying additional 2,6-disubstituted purine compounds useful for inducing dedifferentiation of lineage committed cells into stem cells are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/985,645,filed Nov. 10, 2004, which claims the benefit of U.S. ProvisionalApplication No. 60/518,947, filed Nov. 10, 2003, which are incorporatedherein by reference in their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

Not applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable

BACKGROUND OF THE INVENTION

In mammals, regeneration of injured tissues and limbs is largely limitedby an irreversible differentiation process (see, e.g., Carlson, Dev.Dyn. 226(2):167-81 (2003)). As a consequence, stem cells, in particularembryonic stem cells (ESCs) which can be expanded indefinitely and arepluripotent or multipotent, have attracted considerable attention as atherapeutic approach to the damage caused by cardiovascular disease,neurodegenerative disease and aging (see, e.g., Committee on theBiological and Biomedical Applications of Stem Cell Research, Stem Cellsand the Future of Regenerative Medicine 2002, the National AcademiesPress, Washington, D.C.). However the use of stem cells in cellreplacement therapy remains problematic for a number of reasons,including the lack of a reliable source for stem cells. For example,multipotent human mesenchymal stem cells (MSC) may be isolated from thebone marrow; a large amount of donor bone marrow is required to obtainsufficient quantities of stem cells for most therapeutic or researchapplications.

The ability to dedifferentiate or reverse lineage-committed cells tomultipotent progenitor cells (i.e. multipotent stem cells) overcome manyof these obstacles. With an efficient dedifferentiation process, it isconceivable that healthy, abundant and easily accessible adult cellscould be used to generate different types of functional cells for repairof damaged tissues. Moreover, recent studies of the plasticity of murinemyotubes and other cells derived from adult tissues suggest thatdedifferentiation may be possible in mammalian system (see, e.g.,Odelberg et al., Cell 103:1099-1109 (2000); McGann et al., Proc. Natl.Acad. Sci. USA 98:13699-13704 (2001); and Tsai et al. Developmental Cell2:707-712 (2002)). However, in contrast to the differentiation process,compositions and methods for the control and study of dedifferentiationare lacking.

Cell-based phenotypic assays and, more recently, pathway screens ofsynthetic small molecules and natural products have historicallyprovided very useful chemical probes of complex cellular processes (see,e.g., White, D. J. G. Ed., Proceedings of an International Conference onCyclosporin A (Elsevier, Amsterdam, 1982), 5-19). The identification ofsmall molecules which induce dedifferentiation of mammalian somaticcells should help to elucidate the molecular mechanism of thisphenomenon, and may ultimately allow in vivo tissue regeneration.

Thus, there is a need in the art for compositions and methods forinducing dedifferentiation of lineage committed mammalian cells intomultipotent or pluripotent stem cells. The present invention satisfiesthese and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods forinducing dedifferentiation of lineage committed mammalian cells intomultipotent stem cells.

One embodiment of the present invention provides a compound of FormulaI:

In Formula I, R¹ is a member selected from the group consisting ofhydrogen, C₁₋₄alkyl, C₃₋₈cycloalkyl and C₀₋₂alkylaryl, substituted with0-2 R^(1a) groups that are independently selected from the groupconsisting of halogen, C₁₋₄alkyl, C₁₋₄alkoxy, —N(R^(1b), R^(1b)),—SO₂N(R^(1b), R^(1b)), —C(O)N(R^(1b), R^(1b)) and —O-aryl, or when saidR^(1a) groups are on adjacent ring atoms they are optionally takentogether to form a member selected from the group consisting of—O—(CH₂)₁₋₂—O—, —O—C(CH₃)₂CH₂— and —(CH₂)₃₋₄—, or R¹ is optionally takentogether with the nitrogen to which it is attached to form aheterocycle, optionally substituted with C₁₋₄alkyl, C₃₋₈cycloalkyl,C₁₋₄alkylhydroxy and C₀₋₂alkylaryl; and each R^(1b) group isindependently selected and is a functional group including, but notlimited to, hydrogen and C₁₋₄alkyl.

In Formula I, R² is a functional group including, but not limited to,hydrogen, halogen and -L-R³; L is a functional group including, but notlimited to, —O—, —S— and —NR⁴—, wherein R⁴ is H, or R⁴ is optionallytaken together with R³ and the nitrogen to which both are attached toform a heterocycle, optionally substituted with C₁₋₄ alkyl; R³ is afunctional group including but not limited to, C₁₋₄alkyl, C₃₋₈cycloalkyland aryl-C₀₋₂ alkyl, substituted with 0-2 R^(3a) groups that areindependently selected and are functional groups including, but notlimited to, halogen, C₁₋₄alkyl, C₁₋₄alkoxy, —N(R^(3b), R^(3b)),SO₂N(R^(3b), R^(3b)), C(O)N(R^(3b), R^(3b)) and —O-aryl, or when theR^(3a) groups are on adjacent ring atoms they are optionally takentogether to form a member selected from the group consisting of—O—(CH₂)₁₋₂—O—, —O—C(CH₃)₂CH₂— and —(CH₂)₃₋₄—; and each R^(3b) group isa member that is independently selected and is a functional groupincluding, but not limited to, hydrogen and C₁₋₄alkyl.

The compounds of the present invention include all pharmaceuticallyacceptable salts, isomers, solvates, hydrates and prodrugs thereof.

A further embodiment of the present invention provides a method forinducing dedifferentiation of a lineage committed cell. A lineagecommitted mammalian cell is contacted with a compound of Formula I,whereby the mammalian cell dedifferentiates into a multipotent stemcell. In some embodiments, the method further comprises detectingdedifferentiation of the mammalian cell into a multipotent stem cell(e.g., by detecting loss of expression of a marker gene expressed by thelineage committed mammalian cell). In some embodiments, the lineagecommitted mammalian cell is a myoblast cell.

Even a further embodiment of the present invention provides a method foridentifying compounds that induce dedifferentiation of lineage committedmammalian cells into multipotent stem cells. A lineage committedmammalian cell is contacted with a test compound suspected of inducingdedifferentiation of lineage committed cells. The cells are cultured ina first cell culture media that induces differentiation of themultipotent stem cell into a first cell type and a second cell culturemedia that induces differentiation of the multipotent stem cell into asecond cell type. It is determined whether the cells have undergonedifferentiation into the first or second cell type, wherein induction ofdifferentiation into both the first cell type and the second cell typeidentifies the test compound as a compound that inducesdedifferentiation of lineage committed mammalian cells. In someembodiments of the invention, the first cell culture medium inducesosteogenesis and the second culture medium induces adipogenesis, and thefirst cell type is an osteoblast and the second cell type is anadipocyte. In some embodiments, the test compound is a member selectedfrom the group consisting of: substituted purines (e.g., a 2,6disubstituted purine), pyrimidines, quinazolines, pyrazines,pyrrolopyrimidine, pyrazolopyrimidine, phthalazines, pyridazines, andquinoxalines. In some embodiments, induction of osteogenesis is detectedby detecting expression of an osteogenesis marker gene (e.g., alkalinephosphatase, collagen type I, osteocalcin, ad osteoponin) and inductionof adipogenesis is detected by detecting expression of an adipogenesismarker gene (e.g., ob, Ucp, PPARγ and C/EBPs (see, e.g., Kozak andKozak, Endocrinology 134(2):906-13 (1994) and Lee et al., J. Clin.Invest. 111(4):453-461 (2003).

In yet another embodiment, the present invention provides a method oftreating a bone disorder (e.g., osteoporosis, rickets, osteomalacia,McCune-Albright syndrome, or Paget's disease). A mammalian cell iscontacted with a compound of Formula I, whereby the mammalian celldedifferentiates into a multipotent stem cell. The multipotent stem cellis cultured in a cell culture medium that induces differentiation of themultipotent stem cell into a cell of an osteoblast lineage. In someembodiments, the cell culture medium that induces differentiation of themultipotent stem cell into a cell of an osteoblast lineage comprisesascorbic acid, dexamethasone, and β-glycerophosphate. In someembodiments, the mammalian cell is attached to a solid support (e.g., athree dimensional matrix or a planar surface). In some embodiments,administration is by surgical implantation.

Other embodiments and advantages of the present invention will beapparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the screening method used to identify compounds withdedifferentiation inducing activity.

FIG. 2 illustrates the structure of Compound A.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides compounds, compositions and methods fordedifferentiating lineage committed mammalian cells into stem cells(e.g., multipotent or pluripotent cells). More particularly, the presentinvention provides compounds of Formula I that are useful fordedifferentiating a lineage committed mammalian cells into stem cells.In some embodiments, a composition comprising the compound of Formula Iis provided. In other embodiments, methods of inducing dedifferentiationof lineage committed mammalian cells into stem cells are provided. Inother embodiment, the stem cells are further differentiated into alinage committed cell. In even further embodiments, methods ofidentifying additional compounds useful for inducing dedifferentiaton oflineage committed cells into stem cells are provided.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures for organicand analytical chemistry are those well known and commonly employed inthe art.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, is also meant toinclude those derivatives of alkyl defined in more detail below as“heteroalkyl.” Alkyl groups which are limited to hydrocarbon groups aretermed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—, and further includes those groups described below as“heteroalkylene.” Typically, an alkyl (or alkylene) group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—O—CH₂—CH₂—(CH₃)₂,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified by—CH₂—CH₂—S—CH₂CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from zero to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —CN and —NO₂ in a number ranging from zero to (2m′+1),where m′ is the total number of carbon atoms in such radical. R′, R″ andR′″ each independently refer to hydrogen, unsubstituted (C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl. As used herein, R′, and R″ are fully applicable to R^(3a)and R^(3b). From the above discussion of substituents, one of skill inthe art will understand that the term “alkyl” is meant to include groupssuch as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃,—C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similarly, substituents for the aryl and heteroaryl groups are variedand are selected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′,—NR′—C(O)NR″R′″, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —N₃, —CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, andperfluoro(C₁-C₄)alkyl, in a number ranging from zero to the total numberof open valences on the aromatic ring system; and where R′, R″ and R′″are independently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CH₂)_(r)—B—, wherein A and B are independently —CH₂—, —O—, —NH—,—S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integerof from 1 to 3. One of the single bonds of the new ring so formed mayoptionally be replaced with a double bond. Alternatively, two of thesubstituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CH₂)_(S)—X—(CH₂)_(t)—, where s and t are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituent R′ in —NR′— and —S(O)₂NR′— is selected from hydrogen orunsubstituted (C₁-C₆)alkyl.

The terms “halo” or “halogen” as used herein refer to Cl, Br, F or Isubstituents. The term “haloalkyl”, and the like, refer to an aliphaticcarbon radicals having at least one hydrogen atom replaced by a Cl, Br,F or I atom, including mixtures of different halo atoms. Trihaloalkylincludes trifluoromethyl and the like as preferred radicals, forexample.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N) and sulfur (S).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic,citric, tartaric, methanesulfonic, and the like. Also included are saltsof amino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(3H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

A “lineage committed cell” as used herein, refers to any cell that hasor will differentiate into a particular cell type or related cell types.Lineage committed cells include, for example, osteoblasts, myoblasts,chrondrocytes, and adipocytes.

A “stem cell,” as used herein, refers to any self-renewing pluripotentcell or multipotent cell or progenitor cell or precursor cell that iscapable of differentiating into multiple cell types. Stem cells includethose that are capable of differentiating into cells of osteoblastlineage, a mesenchymal cell lineage (e.g., bone, cartilage, adipose,muscle, stroma, including hematopoietic supportive stroma, and tendon).

“Differentiate” or “differentiation,” as used herein, refers to theprocess by which precursor or progenitor cells (i.e., stem cells)differentiate into specific cell types, e.g., osteoblasts.Differentiated cells can be identified by their patterns of geneexpression and cell surface protein expression. For example, cells of anosteoblast lineage typically express the following genes: alkalinephosphatase, collagen type I, osteocalcin, and osteoponin, and thefollowing bone specific transcription factors: Cbfa1/Runx2, Osx, gsc,Dlx1, Dlx5, Msx1, Cart1, Hoxa1, Hoxa2, Hoxa3, Hoxb1, rae28, Twist, AP-2,Mf1, Pax1, Pax3, Pax9, TBX3, TBX4, TBX5, and Brachyury (see, e.g., Olsenet al, 2000 supra and Nakashima et al., Cell 108(1):17-29 (2002). As afurther example, cells of a myoblast lineage typically express thefollowing genes: MyoD, Myf5, myosin, CD56, and desmin (see, e.g.,Stewart et al., J. Cell Physiol. 196(1):70-8 (2003)). As anotherexample, cells of an adipocyte lineage typically express the followinggenes: ob, Ucp, PPARγ and C/EBPs (see, e.g., Kozak and Kozak,Endocrinology 134(2):906-13 (1994)) and Lee et al., J. Clin. Invest. 111(4): 453-461 (2003).

“Dedifferentiate” or “dedifferentiation,” as used herein, refers to theprocess by which lineage committed cells (e.g., myoblasts orosteoblasts) reverse their lineage commitment and become precursor orprogenitor cells (i.e., multipotent or pluripotent stem cells).Dedifferentiated cells can be identified by loss of patterns of geneexpression and cell surface protein expression associated with thelineage committed cells. For example, myoblasts typically express, interalia, MyoD, Myf5, myosin, CD56 and desmin. A loss of expression ordecrease in expression levels of one or more of these genes indicatesthat a myoblast has undergone dedifferentiation.

“Transdifferentiation” refers to the process refers to the process bywhich precursor or progenitor cells (i.e., stem cells) pre-committed tocell types of one lineage differentiate into specific cell types ofanother lineage, e.g., pre-adipocytes transdifferentiate intoosteoblasts or myoblasts transdifferentiate into osteoblasts.Transdifferentiated cells can be identified by their patterns of geneexpression and cell surface protein expression. Typically, cells of anosteoblast lineage express genes such as, for example, alkalinephosphatase, collagen type I, osteocalcin, and osteoponin; and bonespecific transcription factors such as, for example, Cbfa1/Runx2, Osx,gsc, Dlx1, Dlx5, Msx1, Cart1, Hoxa1, Hoxa2, Hoxa3, Hoxb1, rae28, Twist,AP-2, Mf1, Pax1, Pax3, Pax9, TBX3, TBX4, TBX5, and Brachyury (see, e.g.,Olsen et al, 2000 supra and Nakashima et al., Cell 108(1):17-29 (2002).

“Osteogenesis,” as used herein, refers to proliferation of bone cellsand growth of bone tissue (i.e., synthesis and deposit of new bonematrix). Osteogenesis also refers to differentiation ortransdifferentiation of progenitor or precursor cells into bone cells(i.e., osteoblasts). Progenitor or precursor cells can be pluripotentstem cells such as, e.g., mesenchymal stem cells. Progenitor orprecursor cells can be cells pre-committed to an osteoblast lineage(e.g., pre-osteoblast cells) or cells that are not pre-committed to anosteoblast lineage (e.g., pre-adipocytes or myoblasts).

A “solid support,” as used herein in connection with inducingosteogenesis, refers to a three-dimensional matrix or a planar surfaceon which the stem cells can be cultured. The solid support can bederived from naturally occurring substances (i.e., protein based) orsynthetic substances. For example, matrices based on naturally occurringsubstances may be composed of autologous bone fragments or commerciallyavailable bone substitutes as described in e.g., Clokie et al., J.Craniofac. Surg. 13(1):111-21 (2002) and Isaksson, Swed. Dent. J. Suppl.84:1-46 (1992). Suitable synthetic matrices are described in, e.g., U.S.Pat. Nos. 5,041,138, 5,512,474, and 6,425,222. For example,biodegradable artificial polymers, such as polyglycolic acid,polyorthoester, or polyanhydride can be used for the solid support.Calcium carbonate, aragonite, and porous ceramics (e.g., densehydroxyapatite ceramic) are also suitable for use in the solid support.Polymers such as polypropylene, polyethylene glycol, and polystyrene canalso be used in the solid support. Cells cultured and differentiated ona solid support that is a three-dimensional matrix typically grow on allof the surfaces of the matrix, e.g., internal and external. Cellscultured and differentiated on a solid support that is planar typicallygrow in a monolayer. The term “solid-support” is also used in thecontext of preparing the compounds of Formula I. In this context,“solid-support” refers to a polymeric support, such as a bead, that canbe partially soluble in a suitable solvent or completely insoluble, andis used to bind, for example, a reactant or a reagent of the reaction.Suitable solid-supports include, but are not limited to, PAL resin, Wangresin, and polystyrene resin.

“Culturing,” as used herein, refers to maintaining cells underconditions in which they can proliferate, differentiate, and avoidsenescence. Cells can be cultured in growth media containing appropriategrowth factors.

III. Compounds of the Present Invention and Methods for theirPreparation A. The Compounds of Formula I

In one aspect, the present invention provides compounds of Formula I:

In Formula I, R¹ is a functional group including, but not limited to,hydrogen, C₁₋₄alkyl, C₃₋₈cycloalkyl and C₀₋₂alkylaryl, substituted with0-2 R^(1a) groups that are each independently selected and arefunctional groups including, but not limited to, halogen, C₁₋₄alkyl,C₁₋₄alkoxy, —N(R^(1b), R^(1b)), —SO₂N(R^(1b), R^(1b)), —C(O)N(R^(1b),R^(1b)) and —O-aryl, or when the R^(1a) groups are on adjacent ringatoms they are optionally taken together to form a member selected fromthe group consisting of —O—(CH₂)₁₋₂—O—, —O—C(CH₃)₂CH₂— and —(CH₂)₃₋₄—,or R¹ is optionally taken together with the nitrogen to which it isattached to form a heterocycle, optionally substituted with a functionalgroup including, but not limited to, C₁₋₄alkyl, C₃₋₈cycloalkyl,C₁₋₄alkylhydroxy and C₀₋₂alkylaryl; and each R^(1b) group isindependently selected and is a functional group including, but notlimited to, hydrogen and C₁₋₄alkyl.

In Formula I, R² is a functional group including, but not limited to,hydrogen, halogen and -L-R³; L is a functional group including, but notlimited to, —O—, —S— and —NR⁴—, wherein R⁴ is H, or R⁴ is optionallytaken together with R³ and the nitrogen to which both are attached toform a heterocycle, optionally substituted with C₁₋₄ alkyl; R³ is afunctional group including, but not limited to, C₁₋₄alkyl,C₃₋₈cycloalkyl and aryl-C₀₋₂ alkyl, substituted with 0-2 R^(3a) groupsthat are independently selected and are functional groups including, butnot limited to, halogen, C₁₋₄alkyl, C₁₋₄alkoxy, N(R^(3b), R^(3b)),—SO₂N(R^(3b), R^(3b)), C(O)N(R^(3b), R^(3b)) and —O-aryl, or when theR^(3a) groups are on adjacent ring atoms they are optionally takentogether to form a member selected from the group consisting of—O—(CH₂)₁₋₂—O—, —O—C(CH₃)₂CH₂— and —(CH₂)₃₋₄—; and each R^(3b) group isa member that is independently selected and is a functional groupincluding, but not limited to, hydrogen and C₁₋₄alkyl.

The compounds of the present invention include all pharmaceuticallyacceptable salts, isomers, solvates, hydrates and prodrugs thereof.

In a preferred embodiment, R¹ is a functional group including, but notlimited to, the following:

In a preferred embodiment, R² is a functional group including, but notlimited to, the following:

In some preferred embodiments, R¹ is C₀₋₂alkylaryl. In other preferredembodiments, R¹ is C₀₋₂alkylaryl, substituted with —N(R^(1b), R^(1b)).

In some embodiments, R¹ is

In some embodiments, R² is -L-R³. In some embodiments, L is —NR⁴—,wherein R⁴ is hydrogen, and R³ is C₃₋₈cycloalkyl. In some embodiments,R³ is cyclohexyl.

Preferred compounds of the present invention include but are not limitedto the following compounds:

In a particularly preferred embodiment, the compound has the followingstructure:

The compounds of Formula I can be readily screened for their ability toinduce dedifferentiation of lineage committed mammalian cells (i.e., togenerate stem cells) using the screening methods set forth below and, inparticular, in the examples.

B. Preparation of Compounds

The compounds of the present invention can be prepared by eithersolid-phase or solution-phase synthesis.

1. Solid-Phase Synthesis

Methods directed to the solid-phase synthesis of the compounds ofFormula I are discussed herein in Example I, as well as in Ding et al.,J. Am. Chem. Soc. 124:1594 (2002) and in U.S. patent application Ser.No. 10/687,220, filed on Oct. 15, 2003, U.S. Patent Application No.60/328,763, filed Oct. 12, 2001, U.S. Patent Application No. 60/331,835,filed Nov. 20, 2001, U.S. Patent Application No. 60/346,480, filed Jan.7, 2002, U.S. Patent Application No. 60/348,089, filed Jan. 10, 2002,and U.S. patent application Ser. No. 10/270,030, filed Oct. 12, 2002.

In one aspect, the present invention provides a method for synthesizinga substituted heteroaryl, the method comprising: (a) providing adihaloheteroaryl scaffold moiety; and (b) capturing the dihaloheteroarylscaffold moiety on a resin by nucleophilic substitution of a firsthalogen by a resin-bound amine nucleophile to afford a substitutedheteroaryl, e.g., a resin-bound amine substituted monohaloheteroaryl;(c) reacting the second halogen with a suitably substituted amine oraryl alcohol to afford the resin bound substituted heteroaryl; and (d)cleavage of the substituted heteroaryl from the resin.

Suitable resins useful for the present invention include, but are notlimited to, PAL resin, Wang resin, and polystyrene resin. Other suitableresins would be clear to a person of skill in the art. In a preferredembodiment, the PAL resin is utilized.

In a preferred embodiment, the two halogens, i.e., halo groups, of thedihaloheteroaryl scaffold moiety are independently selected and include,but are not limited to, chloro, fluoro, bromo and iodo. In a presentlypreferred embodiments, the two halogens are chloro groups.

In a preferred embodiment, the method further comprises substitution ofthe second halogen of the dihaloheteroaryl scaffold moiety bynucleophilic displacement or, alternatively, by a coupling reaction. Ina presently preferred embodiment, a coupling reaction is employed tocarry out the substitution of the second halogen of the dihaloheteroarylscaffold moiety. In this connection, the coupling reaction is preferablya palladium-mediated coupling reaction.

It will be readily apparent to those of skill in the art that the twohalogens, i.e., halo groups, of the dihaloheteroaryl scaffold moiety canbe substituted with a number of different functional groups. Suitablefunctional groups include, but are not limited to, anilines, phenols,amines and boronic acids. In a preferred embodiment, the functionalgroups include, but are not limited to, aryl boronic acids, anilines andphenols.

In the compounds of the present invention, N9 is preferablyunsubstituted. If, however, it is desirable to substitute N9, one canperform an initial substitution of N9 prior to substitution of the firsthalogen of the dihaloheteroaryl scaffold moiety. In a preferredembodiment, the initial substitution is carried out using a reactionincluding, but not limited to, alkylation reactions, acylation reactionsand coupling reactions.

Numerous dihaloheteroaryl scaffold moieties can be used in the methodsof the present invention. Examples of suitable dihaloheteroaryl scaffoldmoieties include, but not limited to, purines, pyrimidines,quinazolines, pyrazines, phthalazines, pyradazines and quinoxalines. Inpreferred embodiments of the present invention, the dihaloheteroarylscaffold is a purine.

When a palladium-catalyzed coupling reaction is employed to substitutethe halo groups of the dihaloheteroaryl or the halo group of theresin-bound amine substituted monohaloheteroaryl, thepalladium-catalyzed coupling reaction typically involves reacting thedihaloheteroaryl or the resin-bound amine substituted monohaloheteroarylwith a coupling agent in the presence of a solvent, a palladiumcatalyst, a base and a carbene or phosphine ligand. Suitable couplingagents include, but are not limited to, boronic acids, amines andalcohols. In a presently preferred embodiment, suitable coupling agentsinclude, but are not limited to, aryl boronic acids, anilines andphenols.

In the above methods, carbene or phosphine ligands can be used. Examplesof ligands suitable for use in the methods of the present inventioninclude, but are not limited to, the following carbene and phosphineligands:

In a presently preferred embodiment, the ligand is a phosphine ligandincluding, but not limited to, the following:

A number of bases can be used in carrying out the methods of the presentinvention. Examples of bases suitable for use in the above methodinclude, but are not limited to, cesium carbonate, potassium carbonate,sodium carbonate, sodium bicarbonate, potassium bicarbonate, cesiumbicarbonate, potassium fluoride, potassium phosphate, potassiumtert-butyloxide, sodium tert-butyloxide, and triethylamine.

A number of solvents can be used in carrying out the methods of thepresent invention. Examples of solvents suitable for use in the abovemethod include, but are not limited to, 1,4-dioxane, tetrahydrofuran,dimethoxyethane (DME), dimethylformamide (DMF), benzene and toluene.

A number of palladium catalysts can be used in carrying out the methodsof the present invention. Typically, the oxidation state of thepalladium in the catalyst is (0) or (II). Examples of palladiumcatalysts suitable for use in carrying out the methods of the presentinvention include, but are not limited to, Pd₂(dba)₃, Pd(OAc)₂,Pd(PPh₃)₄, Pd(O), PdCl₂(dppf) and PdCl₂. Such catalysts are known to andused by those of skill in the art and, thus, their structures are known.In a preferred embodiment, the palladium catalyst is Pd₂(dba)₃.

In a preferred embodiment, the foregoing methods further comprisecleaving the compound from the solid support. It will be readilyappreciated that the compounds of the present invention can be readilycleaved from the solid support using standard methods known to and usedby those of skill in the art. Cleavage of a resin-bound compound andliberation of the desired compound from the resin is typically carriedin the presence of an acid. Suitable acids include, but are not limitedto, an organic acid such as formic acid, acetic acid, propionic acid,trichloroacetic acid, trifluoroacetic acid and the like, and inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acid,hydrogen chloride, etc., or the like. The reaction is usually carriedout in a solvent such as water, an alcohol such as methanol, ethanol,1,4, dioxane, methylene chloride, tetrahydrofuran, a mixture thereof orany other solvent which does not adversely influence the reaction.

In yet another aspect of the present invention, the foregoing method isadapted to prepare a library (or an array) of heteroaryl scaffoldmoieties. Typically, the library of substituted scaffold moieties isprepared using a plurality of dihaloheteroaryl scaffold moieties. Assuch, in another aspect, the present invention provides a method forsynthesizing a combinatorial library of substituted heteroaryls (e.g.,heterocycles), the method comprising: providing a plurality ofdihaloheterocyclic scaffold moieties; and capturing thedichloroheterocyclic scaffold moieties on a resin by nucleophilicsubstitution of a first chlorine by a resin-bound amine nucleophile).

In a preferred embodiment, the two halogens, i.e., halo groups, presentin the dihaloheteroaryl scaffold moieties are independently selected andinclude, but are not limited to, chloro, fluoro, bromo and iodo. In apresently preferred embodiments, the two halogens of thedihaloheteroaryl scaffold moieties are chloro groups.

In a preferred embodiment, the method further comprises substitution ofthe second halogen of the dihaloheteroaryl scaffold moieties bynucleophilic displacement or, alternatively, by a coupling reaction. Ina presently preferred embodiment, a coupling reaction is employed tocarry out the substitution of the second halogen of the dihaloheteroarylscaffold moieties. In this connection, the coupling reaction ispreferably a palladium-mediated coupling reaction.

It will be readily apparent to those of skill in the art that the twohalogens, i.e., halo groups, of the dihaloheteroaryl scaffold moietiescan be substituted with a number of different functional groups, each ofwhich is independently selected. Suitable functional groups include, butare not limited to, anilines, phenols, amines and boronic acids (see,Table I). In a presently preferred embodiment, the functional groupsinclude, but are not limited to, aryl boronic acids, anilines andphenols.

If it is desirable to introduce a substitution at N9, the method furthercomprises performing initial substitutions prior to substitution of thefirst halogens of the dihaloheteroaryl scaffold moieties. In a preferredembodiment, the initial substitution is carried out using a reactionincluding, but not limited to, alkylation reactions, acylation reactionsand coupling reactions.

Numerous dihaloheteroaryl scaffold moieties can be used in the methodsof the present invention. Examples of suitable dihaloheteroaryl scaffoldmoieties include, but not limited to, purines, pyrimidines,quinazolines, pyrazines, phthalazines, pyradazines and quinoxalines.

When a palladium-catalyzed coupling reaction is employed to substitutethe halo groups of the dihaloheteroaryl scaffold moieties or the halogroup of the resin-bound amine substituted monohaloheteroaryls, thepalladium-catalyzed coupling reaction typically involves reacting thedihaloheteroaryl or the resin-bound amine substituted monohaloheteroarylwith a coupling agent in the presence of a solvent, a palladiumcatalyst, a base and a carbene or phosphine ligand. Suitable couplingagents include, but are not limited to, boronic acids, amines andalcohols. In a presently preferred embodiment, suitable coupling agentsinclude, but are not limited to, aryl boronic acids, anilines andphenols. It is noted that the foregoing discussions relating to thecarbene or phosphine ligands, bases, solvents, palladium catalysts andcopper catalysts set forth in connection with the methods for preparinga C-2 substituted purine compound are fully applicable to the methodsfor preparing a combinatorial library or array of substituted heteroarylcompound and, thus, they will not be repeated here.

2. Solution-Phase Synthesis

The solution-phase synthesis of the compounds of Formula I involvesfirst substituting 2,6-dihaloheteroaryl with a suitably substitutedamine under appropriate reaction conditions known to one of skill in theart. This is followed by substitution with a suitably substituted amine,aniline or arylalcohol using a Pd catalyst under appropriate reactionconditions known to one of skill in the art. It is noted that theforegoing discussions relating to the carbene or phosphine ligands,bases, solvents and palladium catalysts are set forth with the methodsfor the preparing the compounds of Formula I via solid-support are fullyapplicable to the methods for preparing the compounds of Formula I viasolution phase, and, thus, they will not be repeated here.

IV. Methods for Inducing Cell Dedifferentiation

The compositions of the present invention can conveniently be used inmethods for inducing dedifferentiation of mammalian cells. A mammaliancell is contacted with a compound of Formula I (or a compositionthereof), whereupon the mammalian cell dedifferentiates into amultipotent stem cell.

1. Suitable Cells

Suitable lineage committed mammalian cells can be any lineage committedcell type (e.g., myoblasts or osteoblasts) and can be derived from anysuitable mammal (e.g., rodents such as, for example, mice, rats, guineapigs, and rabbits; non-rodent mammals such as, for example, dogs, cats,pigs, sheep, horses, cows, and goats; primates such as, for example,chimpanzees and humans). The lineage committed cells may be primarycells or may be cells maintained in culture. If the cells are maintainedin culture, they are typically contacted with the compounds/compositionsof the present invention between the 12th and 15th passage in culture.Methods for isolation and culture of human and mammalian cells are wellknown in the art and have been described in, e.g., Humason, ANIMALTISSUE TECHNIQUES, 4^(th) ed., W. H. Freeman and Company (1979);Freshney et al., CULTURE OF ANIMAL CELLS (3rd ed. 1994); andRicciardelli et al., (1989) In Vitro Cell Dev. Biol. 25: 1016.

2. General Culturing Methods

The mammalian cells may be contacted with a compound of Formula I, suchas Compound A, alone or with a compound of Formula I, such as CompoundA, in the presence of growth factors (e.g., fibroblast growth factor orTGF-β). Those of skill in the art will appreciate that the amount of acompound of Formula I, such as Compound A, and growth factors can beadjusted to facilitate dedifferentiation of particular lineage committedcell types. Typically, the amount of Compound A contacted with the cellsis from about 0.1 μM (52 ng/ml) to about 50 μM (2.6 μg/ml), moretypically from about 0.25 μM to about 35 μM, even more typically fromabout 0.5 μM to about 25 μM, yet more typically from about 0.75 μM toabout 15 μM, most typically at about 5 μM.

This aspect of the present invention relies upon routine techniques inthe field of cell culture. Suitable cell culture methods and conditionscan be determined by those of skill in the art using known methodology(see, e.g., Freshney et al., 1994, supra). In general, the cell cultureenvironment includes consideration of such factors as the substrate forcell growth, cell density and cell contract, the gas phase, the medium,and temperature.

Incubation of cells is generally performed under conditions known to beoptimal for cell growth. Such conditions may include, for example, atemperature of approximately 37° C. and a humidified atmospherecontaining approximately 5% CO₂. The duration of the incubation can varywidely, depending on the desired results. In general, incubation ispreferably continued until the cells express suitable Proliferation isconveniently determined using ³H thymidine incorporation or BrdUlabeling.

Plastic dishes, flasks, or roller bottles may be used to culture cellsaccording to the methods of the present invention. Suitable culturevessels include, for example, multi-well plates, petri dishes, tissueculture tubes, flasks, roller bottles, and the like.

Cells are grown at optimal densities that are determined empiricallybased on the cell type. Cells are typically passaged 12-15 times anddiscarded after 15 passages.

Cultured cells are normally grown in an incubator that provides asuitable temperature, e.g., the body temperature of the animal fromwhich is the cells were obtained, accounting for regional variations intemperature. Generally, 37° C. is the preferred temperature for cellculture. Most incubators are humidified to approximately atmosphericconditions.

Important constituents of the gas phase are oxygen and carbon dioxide.Typically, atmospheric oxygen tensions are used for cell cultures.Culture vessels are usually vented into the incubator atmosphere toallow gas exchange by using gas permeable caps or by preventing sealingof the culture vessels. Carbon dioxide plays a role in pH stabilization,along with buffer in the cell media and is typically present at aconcentration of 1-10% in the incubator. The preferred CO₂ concentrationtypically is 5%.

Defined cell media are available as packaged, premixed powders orpresterilized solutions. Examples of commonly used media include MEM-α,DME, RPMI 1640, DMEM, Iscove's complete media, or McCoy's Medium (see,e.g., GibcoBRL/Life Technologies Catalogue and Reference Guide; SigmaCatalogue). Typically, MEM-α or DMEM are used in the methods of theinvention. Defined cell culture media are often supplemented with 5-20%serum, typically heat inactivated serum, e.g., human, horse, calf, andfetal bovine serum. Typically, 10% fetal bovine serum is used in themethods of the invention. The culture medium is usually buffered tomaintain the cells at a pH preferably from about 7.2 to about 7.4. Othersupplements to the media typically include, e.g., antibiotics, aminoacids, and sugars, and growth factors.

B. Methods of Differentiating Dedifferentiated Lineage Committed Cells

One aspect of the present invention provides methods for differentiatingthe multipotent stem cells derived from lineage committed cells. In anexemplary embodiment, lineage committed cells (e.g., myoblasts) arecontacted with a composition comprising Compound A and induced todedifferentiate into multipotent stem cells (e.g., mesenchymal stemcells). The multipotent stem cells are then induced to differentiateinto lineage committed cells. In the case of multipotent mesenchymalstem cells, they are cultured under conditions conducive to inducingdifferentiation into any one of several cell types including, e.g.,osteoblasts, myoblasts and myotubes, and chondrocytes Methods andculture media for differentiating multipotent stem cells into lineagecommitted cells are well known to those of skill in the art and aredescribed in, e.g., U.S. Pat. Nos. 6,617,159; 5,635,386; and 5,397,706.

Differentiation of the multipotent stem cells into differentiated cellscan be detected by any means known in the art including, e.g., detectingexpression of cell type-specific transcription factors, detectingexpression of cell type-specific proteins, and detecting morphologicalchanges in the cells. For example, osteoblasts typically express thefollowing proteins: alkaline phosphatase (ALP), collagen type I,osteocalcin, and osteoponin; and the following transcription factors:Cbfa1/Runx2, gsc, Dlx1, Dlx5, Msx1, Cart1, Hoxa1, Hoxa2, Hoxa3, Hoxb1,rae28, Twist, AP-2, Mf1, Pax1, Pax3, Pax9, TBX3, TBX4, TBX5, andBrachyury (see, e.g., Olsen et al, Annu. Rev. Cell. Dev. Biol. 16:191(2000)). As a further example, myoblasts typically express the followingproteins: MyoD, Myf5, myosin, CD56 and desmin.

1. Detection of Cell-Specific Proteins

Expression of cell-specific proteins may be detected by measuring thelevel of the cell-specific protein or mRNA. One of skill in the art willappreciate that the particular method used to detected the cell-specificprotein or mRNA is not a critical part of the present invention. Methodsof detecting cell-specific proteins and mRNA are well known in the art.For example, the level of particular cell-specific proteins canconveniently be measured using immunoassays such as immunohistochemicalstaining, western blotting, ELISA and the like with an antibody thatselectively binds to the particular cell specific proteins or a fragmentthereof. Detection of the protein using protein-specific antibodies inimmunoassays is known to those of skill in the art (see, e.g., Harlow &Lane, Antibodies: A Laboratory Manual (1988), Coligan, Current Protocolsin Immunology (1991); Goding, Monoclonal Antibodies: Principles andPractice (2d ed. 1986); and Kohler & Milstein, Nature 256:495-497(1975). For measurement of mRNA, amplification, e.g., PCR, LCR, orhybridization assays, e.g., northern hybridization, RNAse protection,dot blotting, are preferred. The level of protein or mRNA is detected,for example, using directly or indirectly labeled detection agents,e.g., fluorescently or radioactively labeled nucleic acids,radioactively or enzymatically labeled antibodies. These assays arewell-known to those of skill in the art and described in, e.g., Ausubel,et al. ed. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (2001). In the case ofcell-specific enzymes, measurement of enzymatic activity (e.g., alkalinephosphatase) can be used as an indicia of cellular differentiation.Methods of measuring cellular enzymes are well known in the art and aredescribed in, e.g., (see, e.g., Harlow & Lane, 1988, supra; Coligan,1991, supra; Goding, 1986, supra; and Kohler & Milstein, 1975, supra).

2. Detection of Cell-Specific Transcription Factors

Expression of cell-specific transcription factors can be detected usingreporter gene assays. These assays are well known to those of skill inthe art and are described in, e.g., Ausebel et al., supra. and U.S.Patent Application No. 60/418,898. For example, expression of the bonespecific transcription factor Cbfa1/Runx2 can be to detect osteogenesis;expression of the chondrocyte specific transcription factors p38 MAPK orc-Maf can be used to detect chondrogenesis; and expression of themyoblast specific transcription factors Mirk (minibrain-relatedkinase)/dyrk1B or FoxO1 can be used to detect myogenesis.

Reporter genes such as, for example, chloramphenicol acetyltransferase,firefly luciferase, bacterial luciferase, or β-galactosidase can be usedin the reporter gene assays. The reporter construct is typicallytransiently or stably transfected into a cell. The promoter region ofthe relevant gene is typically amplified by PCR appropriate primers. Theresulting PCR product is inserted into a suitable cloning vector,amplified and sequenced. The resulting plasmid is digested withappropriate restriction enzymes and the resulting fragment is insertedinto a vector comprising a reporter gene.

For reporter gene assays with transiently transfected cells, the cellsare typically seeded in a 6-well plate at a density of 30,000 cells/wellin 2 mL of growth medium an incubated overnight or for a suitable time.Plasmid DNA is transfected into the cells using a suitable transfectionreagent. After 8 hours, the transfected cells are plated into 96-wellassay plates (e.g., Corning) and treated with an appropriate amount of acompound of Formula I (e.g., Compound A). The cells are incubated for 4days, then the reporter gene activity in the cells is assayed usingmethods known to those of skill in the art.

For reporter gene assays with stably transfected cells, the cells aretypically seeded in a 6-well plate at a density of 30,000 cells/well in2 mL of growth medium an incubated overnight or for a suitable time. Anappropriate amount of reporter plasmid and a vector comprising aselectable marker (e.g., an antibiotic resistance gene) areco-transfected into the cells using an appropriate transfection reagent.After an appropriate incubation time, cells are seeded in a 10 cmculture dish and an appropriate amount of antibiotic is added to theculture medium. Fresh antibiotic is added at appropriate intervals. Theantibiotic resistant colonies are pooled to yield the stably transfectedcells. The transfected cells are plated into 96-well assay plates (e.g.,Corning) and treated with an appropriate amount of a compound of FormulaI (e.g., Compound A). The cells are incubated for 4 days, then thereporter gene activity in the cells is assayed using methods known tothose of skill in the art.

3. Detection of Morphological Changes

Morphological changes in cells are also indicia of cell differentiationand can be detected using any means known to those of skill in the art.Typically, differentiated cells are stained with a suitable dye andmorphological changes are visually detected, e.g., using a microscope.For example, differentiated cells can be stained with Oil Red O, whichidentifies the presence of liquid droplets within the cytoplasmicmembrane characteristic of adipocytes. Methods and compositions forstaining cells to identify particular cell types are well known in theart and are described in, e.g., Albertine and Gee, J. Leuk. Biol.59(5):631-8 (1996); Allsopp et al., J. Immunol. Methods 1998 May;214(1-2):175-86 (1998); Ashley et al., Leuk. Res. 18(1):37-48 (1994);Ashley et al., Leuk. Res. 17(10):873-82 (1993); Boutonnat et al., C. R.Acad. Sci. III 321(11):901-7 (1998); Boyd Cell Growth Differ.4(9):777-84 (1993); Dell'Accio et al., J. Orthop. Res. 21(1):123-31(2003); Ford et al., J. Surg. Res. 62(1):23-8 (1996); Haas et al., ActaHistochem. 102 (3):273-80 (2000); Horan et al., Methods Cell Biol.33:469-90 (1990); Khalaf et al., J. Immunol. Methods 165(1):121-5(1993); Melnicoff et al., J. Leuk. Biol. 43(5):387-97 (1988); Modo etal., Neuroimage 17(2):803-11 (2002); Muirhead, Morphologie 85:27 (2001);Parish, Immunol. Cell Biol. 77(6):499-508 (1999); Pierelli et al.,Methods Cell Biol. 64(1):153-70 (2001); Waters et al., Cytometry48(3):146-52 (2002); Yuan et al., Microvascular Res. 40:228-9 (1990);and U.S. Pat. Nos. 6,387,326; 6,076,583; 5,700,346; 5,318,795;4,792,521; 4,783,401; 4,762,701; and 4,859,584.

V. Methods of Screening

One embodiment of the invention provides a method of screening foradditional compounds that induce dedifferentiation of a lineagecommitted cell. A lineage committed mammalian cell is contacted with atest compound suspected of inducing dedifferentiation of lineagecommitted mammalian cells. Dedifferentiation of the lineage committedcells can be detected by detecting loss of cell-specific proteins andcell-specific transcription factors as described above. To determinewhether the lineage committed cells have dedifferentiated intomultipotent stem cells, the dedifferentiated cells are cultured in atleast two separate cell culture media, each of which inducesdifferentiation of stem cells into different cell types. Assays todetermine whether the dedifferentiated cells have undergonedifferentiation into the first or second cell type are conduction; andinduction of differentiation of the stem cells into cell typesidentifies the test compound as a compound that inducesdedifferentiation of lineage committed mammalian cells.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of potential therapeuticcompounds (candidate compounds). Such “combinatorial chemical libraries”are then screened in one or more assays to identify those librarymembers (particular chemical species or subclasses) that display adesired characteristic activity. The compounds thus identified can serveas conventional “lead compounds” or can themselves be used as potentialor actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biological synthesisby combining a number of chemical “building blocks” such as reagents.Millions of chemical compounds can be synthesized through suchcombinatorial mixing of chemical building blocks (Gallop et al., J. Med.Chem. 37(9):1233-1251 (1994)).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, substituted purines,pyrimidines, quinazolines, pyrazines, pyrrolopyrimidine,pyrazolopyrimidine, phthalazines, pyridazines, and quinoxalines (see,e.g., Ding et al., J. Am. Chem. Soc. 124:1594 (2002); Gray et al.,Science 281:533 (1998); Rosania et al. Nat. Biotechnol. 18:304 (2000);and Rosania et al., Proc. Natl. Acad. Sci. USA. 96:4797 (1999); peptidelibraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res.37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids(PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO93/20242), random bio-oligomers (PCT Publication WO 92/00091),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J.Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses ofsmall compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661(1994)), oligocarbamates (Cho, et al., Science 261:1303 (1993)), and/orpeptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)).See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994),carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522(1996), and U.S. Pat. No. 5,593,853), and small organic moleculelibraries (see, e.g., benzodiazepines, Baum, C&EN, January 18, page 33(1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, U.S. Pat. No. 5,288,514, compounds that regulate adenylcyclase and cyclic AMP, such as, for example, forskolin and itsderivatives, U.S. Pat. Nos. 5,789,439; 5,350,864, and 4,954,642.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.).

A number of well known robotic systems have also been developed forsolution phase chemistries. These systems include automated workstationslike the automated synthesis apparatus developed by Takeda ChemicalIndustries, LTD. (Osaka, Japan) and many robotic systems utilizingrobotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,Hewlett-Packard, Palo Alto, Calif.), which mimic the manual syntheticoperations performed by a chemist. The above devices, with appropriatemodification, are suitable for use with the present invention. Inaddition, numerous combinatorial libraries are themselves commerciallyavailable (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru,Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

The assays to identify compounds that induce dedifferentiation oflineage committed cells are amenable to high throughput screening. Highthroughput assays for evaluating the presence, absence, quantification,or other properties of particular nucleic acids or protein products arewell known to those of skill in the art. Similarly, binding assays andreporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No.5,559,410 discloses high throughput screening methods for proteins, U.S.Pat. No. 5,585,639 discloses high throughput screening methods fornucleic acid binding (i.e., in arrays), while U.S. Pat. Nos. 5,576,220and 5,541,061 disclose high throughput methods of screening forligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.;Precision Systems, Inc., Natick, Mass., etc.). These systems typicallyautomate procedures, including sample and reagent pipeting, liquiddispensing, timed incubations, and final readings of the microplate indetector(s) appropriate for the assay. These configurable systemsprovide high throughput and rapid start up as well as a high degree offlexibility and customization. The manufacturers of such systems providedetailed protocols for various high throughput systems. Thus, e.g.,Zymark Corp. provides technical bulletins describing screening systemsfor detecting the modulation of gene transcription, ligand binding, andthe like.

VI. Methods of Treatment

Another embodiment of the invention provides methods of treatingindividuals with diseases or disorders which can be treated byadministration of differentiated cells. In this embodiment, a lineagemammalian cell is contacted with a compound of Formula I (e.g., CompoundA or a composition thereof), whereupon the mammalian celldedifferentiates into a multipotent stem cell. The multipotent stem cellcan then be cultured under conditions suitable for inducingdifferentiation of the stem cells into a differentiated cell of adesired lineage (e.g., a cell of an osteoblast lineage, a cell of achondrocyte lineage, or a cell of an adipocyte lineage). Thedifferentiated cell is then administered to an individual in need ofsuch treatment. Lineage committed cells can be extracted from thesubject to be treated, i.e., autologous (thereby avoiding immune-basedrejection of the differentiated cells), or can be from a second subject,i.e., heterologous. In either case, administration of cells can becombined with an appropriate immunosuppressive treatment.

1. Administration of Differentiated Cells

Differentiated cells can be administered to a subject by any means knownto those of skill in the art. In an exemplary embodiment of theinvention, differentiated osteoblast cells on an intact solid support(e.g., a three-dimensional matrix or a planar surface) can beadministered to the subject, e.g., via surgical implantation.Alternatively, the differentiated osteoblast cells can be detached fromthe matrix, i.e., by treatment with a protease, before administration tothe subject, e.g., intravenous, subcutaneous, or intraperitoneal.

The cells may be in formulations suitable for administration, such as,for example, aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. Injection solutions and suspensions canbe prepared from sterile powders, granules, and tablets.

For surgical implantation, differentiated cells are typically left on anintact solid support, e.g., a three-dimensional matrix or planarsurface. The matrix or planar surface is surgically implanted into theappropriate site in a subject. For example, a patient needing a bonegraft can have differentiated cells on an intact solid supportsurgically implanted.

In determining the effective amount of the cells to be administered inthe treatment or prophylaxis of conditions owing to diminished oraberrant differentiated cells, the physician evaluates cell toxicity,transplantation reactions, progression of the disease, and theproduction of anti-cell antibodies. For example, osteoblast cellsdifferentiated according to the methods of the present invention can beadministered in an amount effective to provide osteoblasts to thesubject, taking into account the side-effects of the osteoblasts atvarious concentrations, as applied to the mass and overall health of thepatient. Administration can be accomplished via single or divided doses.

One of skill in the art will appreciate that the differentiated cellscan be used alone or in combination with other compounds and therapeuticregimens to induce tissue regeneration (e.g., osteogenesis). In anexemplary embodiment, the stem cells may be induced to differentiateinto osteoblasts which can be administered to a patient in conjunctionwith bone morphogenetic proteins (e.g., BMP-2, BMP-4, and BMP-7) oranti-resorptive medications (e.g., bisphosphonates such as, for example,alendronate sodium and risedronate sodium; hormones, such as, forexample, calcitonin and estrogens, and selective estrogen receptormodulators, such as, for example, raloxifene) that affect boneremodeling cycle. To assess the effect of the administration ofosteoblasts on bone density, a baseline measurement of bone density inan individual who will receive treatment may taken. Bone density isperiodically measured at suitable intervals during and afteradministration of the compounds of Formula I, e.g., Compound A. Methodsand devices for measuring bone density are well known in the art and aredescribed in, e.g., U.S. Pat. Nos. 6,436,042; 6,405,068; 6,320,931;6,302,582; 6,246,745; 6,230,036; 6,213,934; 6,102,567; 6,058,157;5,898,753; 5,891,033; 5,852,647; 5,817,020; 5,782,763; 5,778,045;5,749,363; 5,745,544; 5,715,820; 5,712,892; 5,572,998; and 5,480,439.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Synthesis and Characterization of Compound A

The phosphine ligand for the Pd-catalyzed coupling was purchased fromStrem Chemicals. All the other chemicals were purchased from Aldrich.

Solution Phase Synthesis of 2-(4-morpholinoanilino)-6-cyclohexylpurine(Compound A)

2-(4-Morpholinoanilino)-6-cyclohexylamino-purine (i.e., reversine orCompound A) was synthesized using the methods similar to thosepreviously described in Ding et al., J. Am. Chem. Soc. 124:1594 (2002).To a solution of 2-fluoro-6-chloropurine (87 mg, 0.5 mmol) in n-butanol(5 mL) was added cyclohexylamine (58 μL, 0.5 mmol) anddiisopropylethylamine (100 μL, 0.6 mmol). The mixture was heated to 80°C. with vigorous stirring for 12 hours. The solvent was then removedunder reduced pressure and the crude was used directly in the next stepreaction without further purification. The crude2-fluoro-6-cyclohexylamino-purine (0.5 mmol) was dissolved in ethanol (1mL), followed by addition of 4-morpholinoaniline (178 mg, 1.0 mmol). Themixture was heated to 75° C. in a sealed tube with vigorous stirring for24 hours. The solvent was then removed under reduced pressure and thecrude material was directly purified by flash chromatography to afford2-(4-morpholinoanilino)-6-cyclohexylamino-purine as a pale white solid(130 mg, overall 67% yield). ¹H NMR (500 MHz, DMSO-d6) δ 1.14-1.24 (m,1H), 1.26-1.39 (m, 4H), 1.59-1.67 (m, 1H), 1.73-1.81 (m, 2H), 1.94-1.99(m, 2H), 3.07 (dd, J=4.8, 4.7 Hz, 4H), 3.74 (dd, J=4.8, 4.7 Hz, 4H),3.90-4.06 (m, 2H), 6.93 (d, J=9.1 Hz, 2H), 7.51 (d, J=8.8 Hz, 2H), 8.31(s, 1H), 8.38 (br. s, 1H), 9.68 (br. s, 1H); ¹³C NMR (125 MHz, CD₃OD) δ26.1, 26.5, 33.4, 40.4, 52.1, 67.4, 116.5, 118.6, 124.9, 133.2, 141.8,147.9, 148.6, 152.9, 153.31; MALDI-FTMS for C₂₁H₂₈N₇O (MH⁺): calcd394.2350; found 394.2341.

Example 2 Materials and Methods

Cell culture and small molecule screen. C2C12 cells (ATCC CRL-1772) arecultured in DMEM (Gibco) supplemented with 10% fetal bovine serum(HyClone) at 37° C. in 5% CO₂. The murine C2C12 cell is a myogeniclineage committed myoblast. Upon withdrawal of serum, confluent C2C12cells can differentiate and fuse into characteristic multi-nucleatedmyotubes.

For the small molecule screen, proliferating C2C12 cells are plated intoblack 384-well tissue culture plates (Greiner) at a density of 1,000cells/well in DMEM with 10% FBS. Test compounds (i.e., small molecules)are added at a final concentration of 5 μM after 16 hours (i.e., thetime point when cells typically attach to the bottom of a tissue cultureplate). After the cells are treated with test compounds for four days,the test compound is removed and the cell culture medium is changed toosteogenic differentiation medium (ODM) containing 50 μg/ml ascorbicacid 2-phosphate, 0.1 μM dexamethasone and 10 mM β-glycerophosphate.Cell culture medium is typically changed every two days. After sevenadditional days of culture in ODM, the ODM is removed and cells arelysed by incubation in 10 μL passive lysis buffer (Promega) for 10 min,followed by addition of 10 μL alkaline phosphatase substrate solution(AttoPhos, Promega). After a 15 minute room temperature incubation,fluorescence intensity is read on an Acquest (Molecular Devices) asdirected by the manufacturer.

Osteogenesis Assays. Mammalian cells (e.g., C2C12 myoblast cells) aretreated with a suitable amount of a test compound (e.g., 5 μM reversine)in DMEM supplemented with 10% FBS for four days. The compound is thenremoved and medium is changed to ODM every two days. After seven daysfollowing induction of osteogenesis, the cells are washed with PBS (200μL, 3 times) and fixed with 10% formalin solution (Sigma) for 20 min.The fixed cells are then washed with PBS (200 μL, 3 times) and stainedwith the Alkaline Phosphatase Staining Kit 86R (Sigma) as directed bythe manufacturer. Images are taken on a Nikon Eclipse TE2000 microscopeat 200 fold magnification.

Adipogenesis Assays: Mammalian cells (e.g., C2C12 myoblast cells) weretreated with a suitable amount of a test compound (e.g., 5 μM reversine)in DMEM supplemented with 10% FBS for four days. The compound was thenremoved and medium was changed to adipogenic differentiation medium(ADM) containing 0.5 mM 3-isobutyl-1-methylxanthine, 2.5 μg/ml insulin,and 0.5 μM dexamethasone. Typically, the ADM is replaced every two days.After seven days following induction of adipogenesis, the cells arewashed with PBS (200 μL, 3 times) and fixed with 10% formalin solution(Sigma) for 10 min. The fixed cells are then washed with PBS (200 μL, 3times) and stained with the 0.7% Oil Red O (Sigma) as directed by themanufacturer. Images are taken on a Nikon Eclipse TE2000 microscope at200 fold magnification.

Example 3 Identification of Compound A as a Compound with CellDedifferentiation Inducing Activity

A heterocycle combinatorial library of approximately 50,000 compoundsdesigned around a large number of kinase-directed scaffolds werescreened, including substituted purines, pyrimidines, quinazolines,pyrazines, pyrrolopyrimidine, pyrazolopyrimidine, phthalazines,pyridazines, and quinoxalines was screened to identify small moleculeswith dedifferentiation inducing activity (see, e.g., Ding et al., J. Am.Chem. Soc. 124:1594 (2002); Gray et al., Science 281:533 (1998); Rosaniaet al. Nat. Biotechnol. 18:304 (2000); and Rosania et al., Proc. Natl.Acad. Sci. USA. 96:4797 (1999).

To identify molecules that induce dedifferentiation of mammalian cells,an assay was devised, based on the notion that lineage-reversedmyoblasts should regain multipotency, i.e., they should acquire theability to differentiate into multiple non-permitted cell lineages whenexposed to conditions that typically induce differentiation ofmultipotent mesenchymal progenitor cells into adipocyptes, osteoblastsor chondrocytes. Osteoblast formation was chosen for the primary screensince there are established osteogenic inducing conditions and a highthroughput assay for detecting the bone specific marker, alkalinephosphatase (ALP or ALK) (see, e.g., Wu et al., J. Am. Chem. Soc.124:14520-14521 (2002)).

A two stage screening protocol was used. C2C12 myoblast cells wereinitially treated with the small molecules for four days to inducededifferentiation, and then assayed for their ability to undergoosteogenesis upon addition of known osteogenic inducing agents. To carryout the screen, C2C12 cells were plated in 384-well plates in growthmedium (DMEM with 10% fetal bovine serum) and after overnight incubation(during which time cells attach to the bottom of the plate) 5 μM ofcompound was added. After four days, compound was removed and the mediumwas changed to osteogenic inducing medium (see, e.g., Ding et al., J.Am. Chem. Soc. 124:1594-1596 (2002) containing 50 μg/ml ascorbic acid2-phosphate, 0.1 μM dexamethasone and 10 mM β-glycerophosphate. Theculture was maintained for an additional seven days, cells were lysedand then assayed for ALP activity using the fluorogenic substrate2′-[2′-benzothiazoyl]-6′-hydroxybenzothiazole phosphate (BBTP).

Among a series of 2,6-disubstituted purine analogs identified in theprimary screen, a 2-(4-morpholinoanilino)-6-cyclohexylamino-purineanalog (i.e., Compound A or reversine, FIG. 2) was found to induce thehighest level (7 fold) of ALP activity relative to the DMSO controltreatment. On day four of compound treatment, striking differences wereobserved between the reversine treated and untreated cells. In thecontrol cells (treated only with DMSO), multi-nucleated myotubes wereformed throughout the culture. In contrast, myotube formation wascompletely inhibited in the presence of 5 μM reversine and cellscontinued to grow to form a confluent culture of mononucleated cells. Inaddition, myogenic specific markers such as MyoD and myosin began todisappear. These results suggest that reversine is not simply acting asa selective toxin (see, e.g., Grigoriadis et al., J. Cell Biol.106:2139-2151 (1988)).

Example 4 Cells Gain Multipotency Following Treatment with Reversine

To confirm that the results were not due to transdifferentiation ofmyogenic cells to osteogenic cells, compounds from the primary screenwere tested to determine (1) whether they can induce osteogenesis in theabsence of the osteogenesis inducing cocktail and (2) whether cellstreated with compounds can differentiate into adipocytes underconditions that induce adipogenesis (see, e.g., Jaiswal et al., J. Cell.Biochem. 64:295-312 (1997)). After four days of treatment withreversine, the compound was removed and cells were then grown inosteogenic differentiation medium (ODM) or adipogenic differentiationmedium (ADM). At the end of day seven, under ODM conditions, 35% ofcells stained positive for ALP. Similarly when exposed to ADM condition,40% of cells had the characteristic fat cell morphology, oil dropletsinside the cytoplasmic membrane, and stained positive with Oil Red O.Again, in the control culture, confluent C2C12 cells continue to formmyotubes and were unaffected by the ODM and ADM conditions. Theseresults clearly demonstrate that reversine treated lineage-committedC2C12 myoblasts cells regain multipotency. Moreover, at the effectiveconcentration of reversine (0.5-5 μM), no significant cell death wasobserved.

In addition, transdifferentiation of C2C12 myoblasts to osteoblasts oradipocytes was not observed under the conditions used to induceosteogenesis or adipogenesis. In the absence of ODM, reversine alone hasno osteogenesis activity. Similarly, in the absence of ADM, reversinealone has no adipogenesis activity. These observations confirm thatreversine induces dedifferentiation of C2C12 cells rather thantrandifferentiation to osteogenic lineage. These observations also thatreversine acts as a dedifferentiation inducing agent rather than simplyenriching certain type of progenitor cells by selectively killingmyoblasts.

Example 5 Clonal Analysis of C2C12

Clonal analysis is used to verify that reversine can inducededifferentiation at the single cell level. C2C12 cells were culturedfrom a single cell and treated with reversine. After 6 days oftreatment, each colony was divided into two portions. One portion wascultured in ODM and another portion was cultured in ADM. The cells werethen analyzed using the staining assays described in Example 1.56 of 97colonies were determined to be multipotent.

Example 6 Structure-Activity Analysis of Reversine

A preliminary structure-activity relationship (SAR) analysis of theprimary screen data revealed that both of the N9-H and the NHsubstitution at the C2 position of the purine ring are critical (removalof either can completely abolish activity). However, primary amines atthe C6 position of the purine ring can be replaced with varioushetero-atoms, such as, for example, oxygen and sulfur without loss inactivity, suggesting a H-bond donor at this position is not required.Only a limited group of aromatic substituents can be tolerated at the C2position of the purine ring and an H-bond acceptor is required at thearomatic ring.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A compound of Formula I having the following structure:

wherein: R¹ is a member selected from a group consisting of

R² is a member selected from the group consisting of


2. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier. 